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Abstract

Camera-based optical imaging of the exposed brain allows cortical hemodynamic responses to stimulation to be examined. Typical multispectral imaging systems utilize a camera and illumination at several wavelengths, allowing discrimination between changes in oxy- and deoxyhemoglobin concentration. However, most multispectral imaging systems utilize white light sources and mechanical filter wheels to multiplex illumination wavelengths, which are slow and difficult to synchronize at high frame rates. We present a new LED-based system capable of high-resolution multispectral imaging at frame rates exceeding 220 Hz. This improved performance enables simultaneous visualization of hemoglobin oxygenation dynamics within single vessels, changes in vessel diameters, blood flow dynamics from the motion of erythrocytes, and dynamically changing fluorescence.

Figures (4)

Multispectral imaging system diagram. Multispectral image acquisition begins with a TTL Stimulus Trigger signal sent from the stimulus control computer to the trigger port of the clock generator (red arrow). The 1M60 CCD camera then begins acquiring frames according to the clock signal (dashed red arrow) generated by the clock generator. When the 1M60 acquires frames, it outputs the EXSYNC signal in real-time (black arrow), which is sent to the interrupt ports of the Arduino Diecimila microcontroller. Using a custom strobe function, downsampled strobe signals are generated by the Arduino and sent to the LED drivers which strobe the LEDs (green and blue arrows, example timing diagram shown bottom right). The strobe signals are recorded by the stimulus control computer, along with physiological signals such as blood pressure and ventilation signals from the animal (yellow arrows). System in figure is configured to quantify the concentrations of HbO2, HbR, and HbT.

Gray scale image of exposed rat somatosensory cortex. Images showing concentrations of HbO2, HbR, and HbT at t = 11 seconds (corresponds to dotted line on time course). Time courses showing the average change in HbO2, HbR, and HbT concentration across the entire field of view. Timing of hindpaw stimulus is shown in grey region.

(a) RGB image created using baseline 470 nm and 530 nm images allow veins (blue) and arteries (red) to be easily distinguished. Selected regions are shown in more detail in (b)-(f). (b) Mixing of oxy- and deoxyhemoglobin from different vein branches combining into a single larger vein. (c) Cross sections of vein and artery used for vessel diameter analysis shown in (d). (d) Time courses of vessel diameters. (e) Matrix of line scans showing movement of red blood cells as dark stripes. (f) Time course of blood flow velocity in veins i and ii labeled in (a). Data was collected at 60 fps and averaged across 20 trials. 4 sec stimulus started at t = 6 sec.

(a) Left: Exposed somatosensory cortex under 530 nm illumination. Right: The same field of view under 490 nm illumination (with 500 nm long pass emission filter) showing fluorescence of Oregon Green 488 BAPTA-1 AM calcium indicator. The heterogeneity of the fluorescence signal is due to the discrete nature of the calcium indicator injection sites. (b) Time courses of the selected regions of the cortex indicated by green and blue boxes in (a). Duration of electrical hindpaw stimulation is shown in gray. Inset shows close-up of individual calcium “spikes”. (Note that a signal decrease at 530 nm corresponds to an increase in HbT concentration) (c) Top: Images of the calcium indicator fluorescence during the evolution of the single calcium spike indicated in (b:inset) over a period of 270 ms. Bottom: Images of the change in HbT concentration calculated from the 530 nm reflectance signal showing the evolution of the hemodynamic response over a period of 6 seconds. Data was taken at 30 fps and 25 ms exposure, time averaged across 10 trials.